The invention relates generally to methods of using various chemokine related compositions, more particularly, to methods of treating skin diseases or conditions associated with misregulation of the chemokine MIP-3xcex1, a ligand for the CCR6 chemokine receptor.
The immune system consists of a wide range of distinct cell types, each with important roles to play. See Paul (ed. 1997) Fundamental Immunology 4th ed., Raven Press, New York. The lymphocytes occupy central stage because they are the cells that determine the specificity of immunity, and it is their response that orchestrates the effector limbs of the immune system. Two broad classes of lymphocytes are recognized: the B lymphocytes, which are precursors of antibody secreting cells, and the T (thymus-dependent) lymphocytes. T lymphocytes express important regulatory functions, such as the ability to help or inhibit the development of specific types of immune response, including antibody production and increased microbicidal activity of macrophages. Other T lymphocytes are involved in direct effector functions, such as the lysis of virus infected-cells or certain neoplastic cells.
The chemokines are a large and diverse superfamily of proteins. The superfamily is subdivided into two classical branches, based upon whether the first two cysteines in the chemokine motif are adjacent (termed the xe2x80x9cC-Cxe2x80x9d branch), or spaced by an intervening residue (xe2x80x9cC-X-Cxe2x80x9d). A more recently identified branch of chemokines lacks two cysteines in the corresponding motif, and is represented by the chemokines known as lymphotactins. Another recently identified branch has three intervening residues between the two cysteines, e.g., CX3C chemokines. See, e.g., Schall and Bacon (1994) Current Opinion in Immunology 6:865-873; and Bacon and Schall (1996) Int. Arch. Allergy and Immunol. 109:97-109.
Many factors have been identified which influence the differentiation process of precursor cells, or regulate the physiology or migration properties of specific cell types. These observations indicate that other factors exist whose functions in immune function were heretofore unrecognized. These factors provide for biological activities whose spectra of effects may be distinct from known differentiation or activation factors. The absence of knowledge about the structural, biological, and physiological properties of the regulatory factors which regulate cell physiology in vivo prevents the modulation of the effects of such factors. Thus, medical conditions where regulation of the development or physiology of relevant cells is required remain unmanageable.
The present invention is based, in part, upon the surprising discovery that the MIP-3xcex1 chemokine is expressed in inflamed skin cells. The chemokine is the ligand for the CCR6 receptor. See Greaves, et al. (1997) J. Expt""l Med. 186:837-844. Both the ligand and receptor are expressed at essentially undetectable levels in normal skin, while both are highly upregulated in inflamed skin.
The present invention provides methods of modulating migration of a cell within or to the skin of a mammal comprising administering to the mammal an effective amount of: an antagonist of MIP-3xcex1; an agonist of MIP-3xcex1, and antagonist of CCR6; or an agonist of CCR6. Typically, the migration is within the skin; or may be chemotactic or chemokinetic. In preferred embodiments, the administering is systemic, local, topical, subcutaneous, intracutaneous, or transdermal. Often, the cell is a T cell, B cell, dendritic cell, or dendritic cell precursor. In other embodiments, the cell is a T cell, or moves into the dermal and/or epidermal layers of the skin.
In other embodiments, the administering is of an antagonist of MIP-3xcex1. Generally, the antagonist is selected from: a mutein of natural MIP-3xcex1; an antibody which neutralizes MIP-3xcex1; or an antibody which binds to CCR6. In various embodiments, the mammal is subject to a skin disease or condition, including one selected from cancer, cancer metastasis, skin transplant, or skin graft. Often, the antagonist is administered in combination with an antibiotic, antifungal, antiviral, or analgesic; or may be with an immune suppressive therapeutic, anti-inflammatory drug, growth factor, or immune adjuvant.
In another embodiment, the administering is with a primate MIP-3xcex1. Often, the modulating is attracting the cell, e.g., to a site of cutaneous lesion. The primate MIP-30xcex1 may be administered in combination with an antibiotic, antifungal, antiviral, or analgesic; or with a vasodilator, growth factor, cytokine, anti-inflammatory drug, or immune adjuvant.
Alternatively, the invention provides a method of purifying a population of cells, the method comprising contacting the cells with MIP-3xcex1, thereby resulting in the identification of cells expressing a receptor for MIP-3xcex1. In certain embodiments, the receptor is CCR6, or the contacting results in specific migration of the cells to a site for purification, e.g., through pores of a membrane.
Outline
I. General
II. Chemokine Agonists and Antagonists
A. MIP-3xcex1 and Variants
B. Antibodies
C. Other Molecules
III. Immunoassays
IV. Uses
I. General
The invention is based, in part, on the surprising discovery that the chemokine MIP-3xcex1 has been implicated in roles in skin immunity. In particular, MIP-3xcex1 has been identified as a ligand for the chemokine receptor designated CCR6. Both MIP-3xcex1 and CCR6 expression are undetectable in normal skin, while both are highly upregulated in inflamed skin samples.
The skin consists of a surface layer of epithelium called the epidermis and an underlying layer of connective tissue called the dermis. Under the dermis is a layer which contains large amounts of adipose tissue, the hypodermis. The skin serves a variety of functions, and variations in the character of the dermis and epidermis occur according to functional demands. The appendages of the skin, hair, nails, and sweat and sebaceous glands, are such local specializations of the epidermis. Together, the skin and its appendages form the integument. See, e.g., Fitzpatrick, et al. (eds. 1993) Dermattology in General Medicine 4th ed., McGraw-Hill, NY; Bos (ed. 1989) Skin Immune System CRC Press, Boca Raton, Fla.; Callen (1996) General Practice Dermatology Appleton and Lange; Rook, et al. (eds. 1998) Textbook of Dermatology Blackwell; Habifor and Habie (1995) Clinical Dermatology: A Color Guide to Diagnosis and Therapy Mosby; and Grob (ed. 1997) Epidemiology, Causes and Prevention of Skin Diseases Blackwell.
The epidermis consists of many different cell types in various proportions. The most prevalent cell type is keratinocytes, which make up some 95% of the cells. Cells in the 1-2% range include melanocytes and Langerhans cells. The Langerhans cells are particularly important because they trap antigens that have penetrated the skin, and transport antigens to regional lymph nodes. A small population of xcex3xcex4 T cells can also reside in the epidermis.
The dermis varies in thickness in different regions of the body. It is tough, flexible, and highly elastic, and consists of a feltwork of collagen fibers with abundant elastic fibers. The connective tissue is arranged into deep reticular and superficial papillary layers.
The chemokines are a sub-family of chemoattractant cytokines that were classically characterized by their ability to mediate leukocyte trafficking or migration by binding to specific G-protein-linked seven transmembrane spanning receptors, or GPCRs. Chemokines are divided into four groups based on the primary sequence of the first two cysteines: the CXC, CC, C, and CX3C families. The CXC and C families are effective predominantly on neutrophils and lymphocytes, respectively. The CC chemokines are preferentially effective on macrophages, lymphocytes, and eosinophils.
The chemokine MIP-3xcex1, from human, mouse, and rat, has been described earlier. See, e.g., human, GenBank HSU77035; mouse, GenBank AF099052; rat, GenBank U90447; Li and Adams, WO 94-US9484; and Wilde, et al. WO 9616979; each of which is incorporated herein by reference for all purposes.
A primate, human, MIP-3xcex1 nucleic acid sequence is disclosed in SEQ ID NO: 1 and its corresponding amino acid sequence is disclosed in SEQ ID NO: 2. Of note, the CC motif is present at amino acid residues 6-7. A signal sequence is indicated, but based upon related genes; slightly different processing may occur in different cell types.
A murine, mouse, MIP-3xcex1 chemokine nucleic acid sequence is disclosed in SEQ ID NO: 3 and its corresponding amino acid sequence is disclosed in SEQ ID NO: 4. SignalP software predicts a cleavage between Ala(xe2x88x921) and Ser1; but the actual cleavage may be on either side by a residue or so.
A murine, rat, MIP-3xcex1 chemokine nucleic acid sequence is disclosed in SEQ ID NO: 5 and its corresponding amino acid sequence is disclosed in SEQ ID NO: 6. SignalP software predicts a cleavage between Ala(xe2x88x921) and Ala1; but the actual cleavage may be on either side by a residue or so.
In contrast to naive lymphocytes, memory/effector lymphocytes can access non-lymphoid effector sites and display restricted, often tissue-selective, migration behavior. This results in the presence of such lymphocytes in the peripheral tissues, e.g., outside of the lymphatic and blood volume.
Both human and mouse MIP-3xcex1 are detected in lymph nodes, appendix, PBL, fetal liver, fetal lung, and various cell lines. See, e.g., Rossi, et al. (1997) J. Immunol. 158:1033-1036; Hieshima, et al. (1997) J. Biol. Chem. 272:5846-5853; Baba, et al. (1997) J. Biol. Chem. 272:14893-14898; and Imai, et al. (1997) J. Biol. Chem. 272:15036-15042. The expression in the Langerhans islets suggests a role in skin functions. The data is consistent with MIP-3xcex1 as a product of activated monocytes, and is preferentially expressed in inflamed tissue. This distribution would suggest that MIP-3xcex1 may have a role in attracting memory T cells, and skin dendritic cells (Langerhans cells) and their precursors. These results suggest an important role for MIP-3xcex1 in recruitment of T cells and dendritic cells to peripheral cutaneous sites.
Chemokine receptors are members of the G protein coupled receptor family. See, e.g., Yoshie, et al. (1997) J. Leukoc. Biol. 62:634-644. CCR6 expression has been reported in Greaves, et al. (1997) J. Expt""l Med. 186:837-844; and Liao, et al., (1999) J. Immunol. 162:186-194. Northern blot data showed expression predominantly in the spleen, with lesser amounts in thymus, testis, small intestine, and peripheral blood. Additional transcripts were detected in spleen. Transcripts were not detected in the TF-1, Jurkat, MRC5, JY, and U937 cell lines. Message seems not to be abundantly expressed in the lymphoid lineage, particularly in, e.g., libraries made from cells made from dendritic cell cultures derived from cells selected on the basis of CD1a expression. Expression is lower in DC generated from monocytes.
Another study showed CCR6 was expressed on memory T cells, including most xcex14xcex27 memory cells and cutaneous lymphocyte-associated antigen expressing cells, and on B cells. Chemotaxis of T cells to MIP-3xcex1 was limited to memory cells. See Liao, et al. (1998) J. Immunol. 162:186-194. Antiserum detected CCR6 on CD34+ bone marrow derived dendritic cells.
Having identified the MIP-3xcex1 as a skin related chemokine, it will find use in affecting medical abnormalities of the skin. Common skin disorders involving the immune system include psoriasis, skin cancers, carcinomas, inflammation, allergies, dermatitis, wound healing, infections (both microbial and parasitic), and many others. See, e.g., The Merck Manual, particularly the chapter on dermatologic disorders. These therapeutics may have useful effects on growth or health of appendages of the skin, including, e.g., hair, nails, and sweat and sebaceous glands.
Psoriasis is a chronic inflammatory skin disease that is associated with hyperplastic epidermal keratinocytes and infiltrating mononuclear cells, including T cells, neutrophils and macrophages. Because of this highly mixed inflammatory picture and the resulting complex interrelationships between these different cells, it has been very difficult to dissect the mechanisms that underlie the induction and progression of the disease.
This view of psoriasis also implies that although dormant autoreactive T cells may pre-exist in susceptible individuals, an environmental stimulus is necessary to trigger disease induction. Others believe that the immune system plays only a minor modulatory role in the disease process and that hyperproliferation of keratinocytes is in fact the initiating event in a genetically susceptible host. Research into the pathogenesis of psoriasis has long been hindered by the lack of suitable animal models.
There is growing data indicating that T cells and not keratinocytes are the primary pathogenic component in the disease. The observations herein provide evidence to support the concept that psoriasis-like conditions can indeed result from unregulated T cell responses.
Skin cancers such as basal cell and squamous cell carcinoma are among the most common malignancies. See, e.g., Miller and Maloney (eds. 1997) Cutaneous Oncology: Pathophysiology, Diagnosis, and Management Blackwell; Emmett and Orourke (1991) Malignant Skin Tumours Churchill Livingstone; Friedman. (1990) Cancer of the Skin Saunders. Most of those tumors arise in sun exposed areas of the skin. Immune regulation or clearance of such tumors may depend upon function of the skin immune system. Cells which effect such may be compromised by local misregulation or suppression. The MIP-3xcex1 or antagonists may break a temporary homeostasis which suppresses normal immune response, thereby leading to activation of proper regulatory and immune pathways.
Dermatitis is a superficial inflammation of the skin, characterized by vesicles (when acute), redness, edema, oozing, crusting, scaling, and/or itching. See, e.g., Lepoittevin (ed. 1998) Allergic Contact Dermatitis: The Molecular Basis Springer-Verlag; Rietschel and Fowler (eds. 1995) Fisher""s Contact Dermatitis Lippincott; and Rycroft, et al. (eds. 1994) Textbook of Contact Dermatitis Springer-Verlag. The term eczematous dermatitis is often used to refer to a vesicular dermatitis. Dermatitis may accompany various immune deficiency conditions or diseases, inborn metabolic disorders, or nutritional deficiency diseases. Certain of the symptoms of such conditions may be treated using the present invention.
Pruritus is a sensation that the patient attempts to relieve by scratching. See, e.g., Fleischer and Fleischer (1998) The Clinical Management of Itching: Therapeutic Protocols for Pruritus Parthenon. Many parasitic or infectious conditions may result in those symptoms, which conditions may be cleared by proper reactivation or suppression of immune functions in the skin. Likewise with various allergic or other immune reactions to exposure to various allergic or inflammatory antigens.
II. Chemokine Agonists and Antagonists
Mammalian MIP-3xcex1 chemokines were described previously in WO 98/01557, which describes various migratory assays. Various agonists and antagonists of the natural ligands can be produced. The migration assays may take advantage of the movement of cells through pores in membranes. Chemotaxis may be measured thereby. Alternatively, chemokinetic assays may be developed, which measure the induction of kinetic movement, not necessarily relative to a gradient, per se.
A. MIP-3xcex1 and Variants
MIP-3xcex1 agonists will exhibit some or all of the signaling functions of MIP-3xcex1, e.g., binding, inducing a Ca++ flux, and chemoattracting appropriate receptor bearing cells. Various mammalian MIP-3xcex1 sequences may be evaluated to determine what residues are conserved across species, suggesting what residues may be changed without dramatic effects on biological activity. Alternatively, conservative substitutions are likely to retain biological activity, thus leading to variant forms of the chemokine which will retain agonist activity. Standard methods for screening mutant or variant MIP-3xcex1 polypeptides will determine what sequences will be useful therapeutic agonists.
In addition, certain nucleic acid expression methods may be applied. For example, in skin graft contexts, it may be useful to transfect the grafts with nucleic acids which will be expressed, as appropriate. Various promoters may be operably linked to the gene, thereby allowing for regulated expression. Antisense constructs may prevent expression of the ligand or receptor.
Alternatively, antagonist activity may be tested or screened for. Tests for ability to antagonize chemoattractant activity can be developed using assays as described below. Various ligand homologs can be created which retain receptor binding capacity, but lack signaling capability, thus serving as competitive binding molecules. Small molecules may also be screened for ability to antagonize MIP-3xcex1 function, e.g., chemoattraction, receptor binding, Ca++ flux, and other effects mediated by MIP-3xcex1. See generally Gilman, et al. (eds. 1990) Goodman and Gilman""s: The Pharmacological Bases of Therapeutics, 8th Ed., Pergamon Press; Remington""s Pharmaceutical Sciences, 17th ed. (1990), Mack Publishing Co., Easton, Pa., each of which is incorporated herein by reference.
B. Antibodies
The present invention provides for the use of an antibody or binding composition which specifically binds to MIP-3xcex1, preferably mammalian, e.g., primate, human, cat, dog, rat, or mouse, and neutralizes the ability of the chemokine to mediate its signal. Antibodies can be raised to various MIP-3xcex1 proteins, including individual, polymorphic, allelic, strain, or species variants, and fragments thereof, either in their naturally occurring (full-length) forms or in their recombinant forms. Additionally, antibodies can be raised to MIP-3xcex1 or polypeptides in both their native (or active) forms or in their inactive, e.g., denatured, forms, which may neutralize ligand capacity to mediate its signal. Antibodies may block the interaction of the ligand with its receptor.
Alternatively, receptor antagonists may be produced by making antibodies which bind to the receptor and block ligand binding. With the identification of the CCR6 as a receptor for the cytokine, antibodies to the receptor may be selected for those which block the binding of, or signaling induced by, ligand.
A number of immunogens may be selected to produce antibodies specifically reactive, or selective for binding, with MIP-3xcex1 or CCR6 proteins. Recombinant protein is a preferred immunogen for the production of monoclonal or polyclonal antibodies. Naturally occurring protein, from appropriate sources, e.g., primate, rodent, etc., may also be used either in pure or impure form. Synthetic peptides, made using the MIP-3xcex1 or CCR6 protein sequences described herein, may also be used as an immunogen for the production of antibodies. Recombinant protein can be expressed and purified in eukaryotic or prokaryotic cells as described, e.g., in Coligan, et al. (eds. 1995 and periodic supplements) Current Protocols in Protein Science John Wiley and Sons, New York, N.Y.; and Ausubel, et al (eds. 1987 and periodic supplements) Current Protocols in Molecular Biology, Greene/Wiley, New York, N.Y. Naturally folded or denatured material, perhaps expressed on cell surfaces, can be used, as appropriate, for producing antibodies. Either monoclonal or polyclonal antibodies may be generated, e.g., for subsequent use in immunoassays to measure the protein, or for immunopurification methods.
Methods of producing polyclonal antibodies are well known to those of skill in the art. Typically, an immunogen, preferably a purified protein, is mixed with an adjuvant and animals are immunized with the mixture. The animal""s immune response to the immunogen preparation is monitored by taking test bleeds and determining the titer of reactivity to, e.g., the MIP-3xcex1, protein or polypeptide of interest. For example, when appropriately high titers of antibody to the immunogen are obtained, usually after repeated immunizations, blood is collected from the animal and antisera are prepared. Further fractionation of the antisera to enrich for antibodies reactive to the protein can be performed, if desired. See, e.g., Harlow and Lane Antibodies, A Laboratory Manual; or Coligan (ed.) Current Protocols in Immunology. Immunization can also be performed through other methods, e.g., DNA vector immunization. See, e.g., Wang, et al. (1997) Virology 228:278-284. Affinity purification, or absorptions, can be used to select for desired specificity of binding.
Monoclonal antibodies may be obtained by various techniques familiar to those skilled in the art. Typically, spleen cells from an animal immunized with a desired antigen are immortalized, commonly by fusion with a myeloma cell. See, Kohler and Milstein (1976) Eur. J. Immunol. 6:511-519. Alternative methods of immortalization include transformation with Epstein Barr Virus, oncogenes, or retroviruses, or other methods known in the art. See, e.g., Doyle, et al. (eds. 1994 and periodic supplements) Cell and Tissue Culture: Laboratory Procedures, John Wiley and Sons, New York, N.Y. Colonies arising from single immortalized cells are screened for production of antibodies of the desired specificity and affinity for the antigen, and yield of the monoclonal antibodies produced by such cells may be enhanced by various techniques, including injection into the peritoneal cavity of a vertebrate host. Alternatively, one may isolate DNA sequences which encode a monoclonal antibody or a binding fragment thereof by screening a DNA library from human B cells according, e.g., to the general protocol outlined by Huse, et al. (1989) Science 246:1275-1281.
Antibodies or binding compositions, including binding fragments and single chain versions, against predetermined fragments of MIP-3xcex1 or CCR6 polypeptides can be raised by immunization of animals with conjugates of the fragments with carrier proteins as described above. Monoclonal antibodies are prepared from cells secreting the desired antibody. These antibodies can be screened for binding to normal or defective MIP-3xcex1 protein, or screened for capacity to block cell MIP-3xcex1 mediated chemoattraction or chemokinetic activity. These monoclonal antibodies will usually bind with at least a KD of about 1 mM, more usually at least about 300 xcexcM, typically at least about 10 xcexcM, more typically at least about 30 xcexcM, preferably at least about 10 xcexcM, and more preferably at least about 3 xcexcM or better.
In some instances, it is desirable to prepare monoclonal antibodies (mAbs) from various mammalian hosts, such as mice, rodents, primates, humans, etc. Description of techniques for preparing such monoclonal antibodies may be found in, e.g., Stites, et al. (eds.) Basic and Clinical Immunology (4th ed.) Lange Medical Publications, Los Altos, Calif., and references cited therein; Harlow and Lane (1988) Antibodies: A Laboratory Manual CSH Press; Goding (1986) Monoclonal Antibodies: Principles and Practice (2d ed.) Academic Press, New York, N.Y.; and particularly in Kohler and Milstein (1975) Nature 256:495-497, which discusses one method of generating monoclonal antibodies. Summarized briefly, this method involves injecting an animal with an immunogen. The animal is then sacrificed and cells taken from its spleen, which are then fused with myeloma cells. The result is a hybrid cell or xe2x80x9chybridomaxe2x80x9d that is capable of reproducing in vitro. The population of hybridomas is then screened to isolate individual clones, each of which secrete a single antibody species to the immunogen. In this manner, the individual antibody species obtained are the products of immortalized and cloned single B cells from the immune animal generated in response to a specific site recognized on the immunogenic substance.
Other suitable techniques involve selection of libraries of antibodies in phage or similar vectors. See, e.g., Huse, et al. (1989) xe2x80x9cGeneration of a Large Combinatorial Library of the Immunoglobulin Repertoire in Phage Lambda,xe2x80x9d Science 246:1275-1281; and Ward, et al. (1989) Nature 341:544-546. The polypeptides and antibodies of the present invention may be used with or without modification, including chimeric or humanized antibodies. Frequently, the polypeptides and antibodies will be labeled by joining, either covalently or non-covalently, a substance which provides for a detectable signal. A wide variety of labels and conjugation techniques are known and are reported extensively in both the scientific and patent literature. Suitable labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent moieties, chemiluminescent moieties, magnetic particles, and the like. Patents teaching the use of such labels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241. Also, recombinant immunoglobulins may be produced, see, Cabilly, U.S. Pat. No. 4,816,567; and Queen, et al. (1989) Proc. Nat""l Acad. Sci. USA 86:10029-10033; or made in transgenic mice, see Mendez, et al. (1997) Nature Genetics 15:146-156.
Antibody binding compounds, including binding fragments, of this invention can have significant diagnostic or therapeutic value. They can be useful as non-neutralizing binding compounds and can be coupled to toxins or radionuclides so that when the binding compound binds to the antigen, a cell expressing it, e.g., on its surface, is killed. Further, these binding compounds can be conjugated to drugs or other therapeutic agents, either directly or indirectly by means of a linker, and may effect drug targeting.
C. Other Molecules
Antibodies are merely one form of specific binding compositions. Other binding compositions, which will often have similar uses, include molecules that bind with specificity to MIP-3xcex1 receptor, e.g., CCR6, in a binding partner-binding partner fashion, an antibody-antigen interaction, or in a natural physiologically relevant protein-protein interaction, either covalent or non-covalent, e.g., proteins which specifically associate with MIP-3xcex1 receptor protein. The molecule may be a polymer, or chemical reagent. A functional analog may be a protein with structural modifications, or may be a structurally unrelated molecule, e.g., which has a molecular shape which interacts with the appropriate binding determinants. Application of, e.g., Systematic Evolution of Ligand by Exponential Enrichment (SELEX) technology, methods are available to select specific binding constructs for desired targets. See, e.g., Colas, et al. (1996) Nature 380:548-550; Cohen, et al. (1998) Proc. Nat""l Acad. Sci. USA 95:14272-14277; Kolonin, et al. (1998) Proc. Nat""l Acad. Sci. USA 95:14266-14271; Famulok, et al. (1998) Curr. Opin. Chem. Biol. 2:320-327; and Eaton, et al. (1997) Bioorg. Med. Chem. 5:1087-1096.
Drug screening using antibodies or MIP-3xcex1 or fragments thereof can be performed to identify compounds having binding affinity to MIP-3xcex1, or can block or simulate the natural interaction with ligand. Subsequent biological assays can then be utilized to determine if the compound has intrinsic blocking activity and is therefore an antagonist. Likewise, a compound having intrinsic stimulating activity can signal to the cells via the MIP-3xcex1 pathway and is thus an agonist in that it simulates the activity of a ligand. Mutein antagonists may be developed which maintain receptor binding but lack signaling.
Structural studies of the ligands will lead to design of new variants, particularly analogs exhibiting agonist or antagonist properties on the receptor. This can be combined with previously described screening methods to isolate muteins exhibiting desired spectra of activities.
As receptor specific binding molecules are provided, also included are small molecules identified by screening procedures. In particular, it is well known in the art how to screen for small molecules which interfere, e.g., with ligand binding to the receptor, often by specific binding to the receptor and blocking of binding by natural ligand. See, e.g., meetings on High Throughput Screening, International Business Communications, Southborough, Mass. 01772-1749. Such molecules may compete with natural ligands, and selectively bind to the MIP-3xcex1 or CCR6
III. Immunoassays
Immunoassays are valuable in diagnosing a disease or disorder associated with MIP-3xcex1 imbalance or pathology. Qualitative or quantitative measurement of a particular protein can be performed by a variety of immunoassay methods. For a review of immunological and immunoassay procedures in general, see Stites and Terr (eds. 1991) Basic and Clinical Immunology (7th ed.). Moreover, the immunoassays of the present invention can be performed in many configurations, which are reviewed extensively in, e.g., Maggio (ed. 1980) Enzyme Immunoassay CRC Press, Boca Raton, Fla.; Tijan (1985) xe2x80x9cPractice and Theory of Enzyme Immunoassays,xe2x80x9d Laboratory Techniques in Biochemistry and Molecular Biology, Elsevier Science Publishers B.V., Amsterdam; Harlow and Lane Antibodies: A Laboratory Manual, supra; Chan (ed. 1987) Immunoassay: A Practical Guide Academic Press, Orlando, Fla.; Price and Newman (eds. 1991) Principles and Practice of Immunoassays Stockton Press, NY; and Ngo (ed. 1988) Non-isotopic Immunoassays Plenum Press, NY.
In particular, the present invention provides various skin related diseases as conditions susceptible to analysis or diagnosis by evaluating MIP-3xcex1 and/or CCR6. For example, the likelihood of skin rejection in a graft situation would be evaluated by the numbers or types of MIP-3xcex1 or CCR6 bearing cells present. Prophylactic downregulation may be useful to prevent the recruitment of dermal T or NK cells. Response to various skin tumors may be evaluated by the presence or absence of MIP-3xcex1 and/or CCR6 bearing cells.
Immunoassays for measurement of MIP-3xcex1 proteins or peptides can be performed by a variety of methods known to those skilled in the art. In brief, immunoassays to measure the protein can be either competitive or noncompetitive binding assays. In competitive binding assays, the sample to be analyzed competes with a labeled analyte for specific binding sites on a capture agent bound to a solid surface. Preferably the capture agent is an antibody specifically reactive with MIP-3xcex1 proteins produced as described above. The concentration of labeled analyte bound to the capture agent is inversely proportional to the amount of free analyte present in the sample.
In a competitive binding immunoassay, typically the MIP-3xcex1 protein present in the sample competes with labeled protein for binding to a specific binding agent, e.g., an antibody specifically reactive with the MIP-3xcex1 protein. The binding agent may be bound to a solid substrate or surface to effect separation of bound labeled protein from the unbound labeled protein. Alternately, the competitive binding assay may be conducted in liquid phase and a variety of techniques known in the art may be used to separate the bound labeled protein from the unbound labeled protein. Following separation, the amount of bound labeled protein is determined. The amount of protein present in the sample is inversely proportional to the amount of labeled protein binding.
Alternatively, a homogeneous immunoassay may be performed in which a separation step is not needed. In these immunoassays, the label on the protein is altered by the binding of the protein to its specific binding agent. This alteration in the labeled protein results in a decrease or increase in the signal emitted by label, so that measurement of the label at the end of the immunoassay allows for detection or quantitation of the protein.
MIP-3xcex1 proteins may also be determined by a variety of noncompetitive immunoassay methods. For example, a two-site, solid phase sandwich immunoassay may be used. In this type of assay, a binding agent for the protein, e.g., an antibody, is attached to a solid support. A second protein binding agent, which may also be an antibody, and which binds the protein at a different site, is labeled. After binding at both sites on the protein has occurred, the unbound labeled binding agent is removed and the amount of labeled binding agent bound to the solid phase is measured. The amount of labeled binding agent bound is directly proportional to the amount of protein in the sample.
Western blot analysis can be used to determine the presence of MIP-3xcex1 or CCR6 proteins in a sample. Electrophoresis is carried out, for example, on a tissue sample suspected of containing the protein. Following electrophoresis to separate the proteins, and transfer of the proteins to a suitable solid support, e.g., a nitrocellulose filter, the solid support is incubated with an antibody reactive with the protein. This antibody may be labeled, or alternatively may be detected by subsequent incubation with a second labeled antibody that binds the primary antibody.
The immunoassay formats described above may employ labeled assay components. The label may be coupled directly or indirectly to the desired component of the assay according to methods well known in the art. A wide variety of labels and methods may be used. Traditionally, a radioactive label incorporating 3H, 125I, 35S, 14C, or 32P was used. Non-radioactive labels include ligands which bind to labeled antibodies, fluorophores, chemiluminescent agents, enzymes, and antibodies which can serve as specific binding pair members for a labeled ligand. The choice of label depends on sensitivity required, ease of conjugation with the compound, stability requirements, and available instrumentation. For a review of various labeling or signal producing systems which may be used, see U.S. Pat. No. 4,391,904.
Antibodies reactive with a particular protein can also be measured by a variety of immunoassay methods. Thus modifications of the above procedures may be used to determine the amounts or affinities of various MIP-3xcex1 or CCR6 antibodies or antibody preparations. For a review of immunological and immunoassay procedures applicable to the measurement of antibodies by immunoassay techniques, see, e.g., Stites and Terr (eds.) Basic and Clinical Immunology (7th ed.) supra; Maggio (ed.) Enzyme Immunoassay, supra; and Harlow and Lane Antibodies, A Laboratory Manual, supra.
Screens to evaluate the binding and activity of mAbs and binding compositions encompass a variety of methods. Binding can be assayed by detectably labeling the antibody or binding composition as described above. Cells responsive to MIP-3xcex1 can be used to assay antibody or binding composition.
To evaluate MIP-3xcex1 chemoattraction or chemokinetic ability, experimental animals, e.g., mice, are preferably used. Skin, e.g., Langerhans, cell counts are made prior to and at various time points after administration of a bolus of the candidate agonist or antagonist. Levels are analyzed in various samples, e.g., blood, serum, nasal or pulmonary lavages, or tissue biopsy staining. A successful depleting mAb or binding composition will significantly lower the level of CCR6 bearing cells. Such may be at least about 10%, preferably at least about 20%, 30%, 50%, 70%, or more.
Evaluation of antibodies can be performed in other animals, e.g., humans using various methods. For example, blood samples are withdrawn from patients suffering from a skin related disease or disorder before and after treatment with a candidate mAb.
IV. Uses
The exquisite tissue-selective homing of lymphocytes has long been appreciated as central for the control of systemic immune responses. Recent advances in the field support a model in which leukocyte homing is achieved by sequential engagement of differentially expressed and independently regulated vascular and leukocyte adhesion molecules, and signaling receptors and their ligands. Butcher and Picker (1996) Science 272:60-66. The observation that chemokines, a superfamily of small secreted proteins with G protein-coupled receptors (Baggiolini (1998) Nature 392:565-568) can attract leukocytes led to the hypothesis that chemokines provide key signals directing recruitment of T lymphocyte subsets into lymphoid and extra-lymphoid immune effector sites. The inflamed skin-specific expression of MIP-3xcex1 and CCR6 suggests that such skin-specific chemokines selectively attract functional subsets of lymphocytes into the skin.
As such, the present invention provides means to purify desired skin cell subsets. The chemoattractive or chemokinetic effects on those cells can be the basis of purification methods. Methods exist for selective migration and recovery of cells to or from the chemokine, e.g., through porous membrane, or to various locations in a culture. Other methods exist to selectively separate cells of particular shapes from others. Alternatively, labeling can be used to FACS sort cells which specifically bind the chemokine. Populations of substantially homogeneous Langerhans or skin derived cells will have important utility in research or therapeutic environments.
While MIP-3xcex1 is likely to have functional effects on CCR6 bearing subsets of cells, e.g., T and B cells and precursors, other cells which may also be responsive include dendritic cells or granulocytes, e.g., neutrophils and/or eosinophils, or their precursors. Effects on various cell types may be indirect, as well as direct. A statistically significant change in the numbers of cells will typically be at least about 10%, preferably 20%, 30%, 50%, 70%, 90%, or more. Effects of greater than 100%, e.g., 130%, 150%, 2X, 3X, 5X, etc., will often be desired. The effects may be specific in causing chemotaxis to specific points, or may be chemokinetic, in inducing general movement of cells, but not necessarily in a specific direction, e.g., of concentration gradient.
The present invention will be useful in the treatment of medical conditions or diseases associated with immunological conditions of the skin. See, e.g., Bos (ed. 1990) Skin Immune System CRC Press, Boca Raton, Fla.; Fitzpatrick, et al. (eds. 1993) Dermatology in General Medicine (4th ed.) McGraw-Hill, NY.; Rook, et al. (eds. 1998) Textbook of Dermatology Blackwell; Habifor and Habie (1995) Clinical Dermatology: A Color Guide to Diagnosis and Therapy Mosby; Grob (ed. 1997) Epidemiology, Causes and Prevention of Skin Diseases Blackwell; Frank, et al. (eds. 1995) Samter""s Immunologic Diseases, 5th Ed., vols. I-II, Little, Brown and Co., Boston, Mass.; Coffman, et al (1989) Science 245:308-310; and Frick, et al. (1988) J. Allergy Clin. Immunol. 82:199-225. The agonists or antagonists described may be combined with other treatments of the medical conditions described herein, e.g., an antibiotic, antifungal, antiviral, immune suppressive therapeutic, immune adjuvant, analgesic, anti-inflammatory drug, growth factor, cytokine, vasodilator, or vasoconstrictor. See, e.g, the Physician""s Desk Reference, both prescription and non-prescription compendiums.
The CCR6 receptor appears to be preferentially expressed on CD4+ memory T cells. Its ligand, MIP-3xcex1, is an inflammatory chemokine expressed by cellular constituents of the skin, whose expression is inducible after stimulation with T cell-derived proinflammatory meidators such as IFN-xcex3 and IL-17. Thus, CD4+ memory T cell mediated skin conditions are therapeutic targets of the antagonists, e.g., psoriasis, atopic dermatitis, contact dermatitis, SLE, and lichen ruber planus.
Preferred combination therapies include the MIP-3xcex1 reagent with various anti-inflammatory agents, such as topical, transdermal, or systemic steroids or corticosteroids. Systemic, topical, transdermal, or systemic retinoid or retinoid-like compounds, or vitamin D analogs, may be administered with the MIP-3xcex1 therapeutics. Alternatively, various forms of UV light may be used in combination with these therapeutics, e.g., ultraviolet A, ultraviolet B, or narrow bands of UVB.
For example, the MIP-3xcex1 ligands would be expected to signal specifically to the cell types expressing their receptor. Thus, it will be possible to block signaling, e.g., to the T cell or B cell subsets, by reagents which block receptor signaling, e.g., antibodies to ligand, and small drug antagonists.
Standard immunological techniques are described, e.g., in Hertzenberg, et al. (eds. 1996) Weir""s Handbook of Experimental Immunology vols. 1-4, Blackwell Science; Coligan (1991) Current Protocols in Immunology Wiley/Greene, NY; and Methods in Enzymology volumes 70, 73, 74, 84, 92, 93, 108, 116, 121, 132, 150, 162, and 163. These will allow use of the reagents for purifying cell subpopulations, etc.
To prepare pharmaceutical or sterile compositions including, e.g., MIP-3xcex1, the material is admixed with a pharmaceutically acceptable carrier or excipient which is preferably inert. Preparation of such pharmaceutical compositions is known in the art, see, e.g., Remington""s Pharmaceutical Sciences and U.S. Pharmacopeia: National Formulary, Mack Publishing Company, Easton, Pa. (1984). Typically, therapeutic compositions are sterile. Alternatively, MIP-3xcex1 antagonist compositions can be prepared.
Agonists, e.g., natural ligand, or antagonists, e.g., antibodies or binding compositions, are normally administered parenterally, preferably intravenously. Since such protein or peptide antagonists may be immunogenic they are preferably administered slowly, either by a conventional IV administration set or from a subcutaneous depot, e.g. as taught by Tomasi, et al., U.S. Pat. No. 4,732,863. However, as a skin target, the administration may be topical, transdermal, intradermal, subcutaneous, or even systemic.
When administered parenterally the therapeutics will be formulated in a unit dosage injectable form (solution, suspension, emulsion) in association with a pharmaceutically acceptable parenteral vehicle. Such vehicles are inherently nontoxic and nontherapeutic. The antagonist may be administered in aqueous vehicles such as water, saline, or buffered vehicles with or without various additives and/or diluting agents. Alternatively, a suspension, such as a zinc suspension, can be prepared to include the peptide. Such a suspension can be useful for subcutaneous (SQ), intradermal (ID), or intramuscular (IM) injection. The proportion of therapeutic entity and additive can be varied over a broad range so long as both are present in effective amounts. The therapeutic is preferably formulated in purified form substantially free of aggregates, other proteins, endotoxins, and the like, at concentrations of about 5 to 30 mg/ml, preferably 10 to 20 mg/ml. Preferably, the endotoxin levels are less than 2.5 EU/ml. See, e.g., Avis, et al. (eds. 1993) Pharmaceutical Dosage Forms: Parenteral Medications 2d ed., Dekker, NY; Lieberman, et al. (eds. 1990) Pharmaceutical Dosage Forms: Tablets 2d ed., Dekker, NY; Lieberman, et al. (eds. 1990) Pharmaceutical Dosage Forms: Disperse Systems Dekker, NY; Fodor, et al. (1991) Science 251:767-773; Coligan (ed.) Current Protocols in Immunology; Hood, et al. Immunology Benjamin/Cummings; Paul (ed. 1997) Fundamental Immunology 4th ed., Academic Press; Parce, et al. (1989) Science 246:243-247; Owicki, et al. (1990) Proc. Nat""l Acad. Sci. USA 87:4007-4011; and Blundell and Johnson (1976) Protein Crystallography, Academic Press, New York. Local, e.g., topical or transdermal, administration will often be particularly useful.
Selecting an administration regimen for a therapeutic agonist or antagonist depends on several factors, including the serum or tissue turnover rate of the therapeutic, the immunogenicity of the therapeutic, or the accessibility of the target cells. Preferably, an administration regimen maximizes the amount of therapeutic delivered to the patient consistent with an acceptable level of side effects. Accordingly, the amount of therapeutic delivered depends in part on the particular agonist or antagonist and the severity of the condition being treated. Guidance in selecting appropriate doses of antibodies is found in the literature on therapeutic uses, e.g. Bach et al., chapter 22, in Ferrone, et al. (eds. 1985) Handbook of Monoclonal Antibodies Noges Publications, Park Ridge, N.J.; and Russell, pgs. 303-357, and Smith et al., pgs. 365-389, in Haber, et al. (eds. 1977) Antibodies in Human Diagnosis and Therapy Raven Press, New York, N.Y.
Determination of the appropriate dose is made by the clinician, e.g., using parameters or factors known in the art to affect treatment or predicted to affect treatment. Generally, the dose begins with an amount somewhat less than the optimum dose and it is increased by small increments thereafter until the desired or optimum effect is achieved relative to any negative side effects. Numbers of CCR6 bearing cells in defined samples might be important indicators of when an effective dose is reached. Preferably, an antibody or binding composition thereof that will be used is derived from the same species as the animal targeted for treatment, thereby minimizing a humoral response to the reagent.
The total weekly dose ranges for antibodies or fragments thereof, which specifically bind to MIP-3xcex1, range generally from about 1 ng, more generally from about 10 ng, typically from about 100 ng; more typically from about 1 xcexcg, more typically from about 10 xcexcg, preferably from about 100 xcexcg, and more preferably from about 1 mg per kilogram body weight. Although higher amounts may be more efficacious, the lower doses typically will have fewer adverse effects. Generally the range will be less than 100 mg, preferably less than about 50 mg, and more preferably less than about 25 mg per kilogram body weight.
The weekly dose ranges for antagonists, e.g., antibody, binding fragments, range from about 10 xcexcg, preferably at least about 50 xcexcg, and more preferably at least about 100 xcexcg per kilogram of body weight. Generally, the range will be less than about 1000xcexcg, preferably less than about 500 xcexcg, and more preferably less than about 100 xcexcg per kilogram of body weight. Dosages are on a schedule which effects the desired treatment and can be periodic over shorter or longer term. In general, ranges will be from at least about 10 xcexcg to about 50 mg, preferably about 100 xcexcg to about 10 mg per kilogram body weight.
Other antagonists of the ligands, e.g., muteins, are also contemplated. Hourly dose ranges for muteins range from at least about 10 xcexcg, generally at least about 50 xcexcg, typically at least about 100 xcexcg, and preferably at least 500 xcexcg per hour. Generally the dosage will be less than about 100 mg, typically less than about 30 mg, preferably less than about 10 mg, and more preferably less than about 6 mg per hour. General ranges will be from at least about 1xcexcg to about 1000 xcexcg, preferably about 10 xcexcg to about 500 xcexcg per hour.
In particular contexts, e.g., transplant or skin grafts, may involve the administration of the therapeutics in different forms. For example, in a skin graft, the tissue may be immersed in a sterile medium containing the therapeutic resulting in a prophylactic effect on cell migration soon after the graft is applied.
The present invention also provides for administration of MIP-3xcex1 antibodies or binding compositions in combination with known therapies, e.g., steroids, particularly glucocorticoids, which alleviate the symptoms associated with excessive inflammatory responses. Daily dosages for glucocorticoids will range from at least about 1 mg, generally at least about 2 mg, and preferably at least about 5 mg per day. Generally, the dosage will be less than about 100 mg, typically less than about 50 mg, preferably less than about 20 mg, and more preferably at least about 10 mg per day. In general, the ranges will be from at least about 1 mg to about 100 mg, preferably from about 2 mg to 50 mg per day.
The phrase xe2x80x9ceffective amountxe2x80x9d means an amount sufficient to effect a desired response, or to ameliorate a symptom or sign of the skin condition. Typical mammalian hosts will include mice, rats, cats, dogs, and primates, including humans. An effective amount for a particular patient may vary depending on factors such as the condition being treated, the overall health of the patient, the method, route, and dose of administration and the severity of side affects. Preferably, the effect will result in a change in quantitation of at least about 10%, preferably at least 20%, 30%, 50%, 70%, or even 90% or more. When in combination, an effective amount is in ratio to a combination of components and the effect is not limited to individual components alone.
An effective amount of therapeutic will modulate the symptoms typically by at least about 10%; usually by at least about 20%; preferably at least about 30%; or more preferably at least about 50%. Alternatively, modulation of migration will mean that the migration or trafficking of various cell types is affected. Such will result in, e.g., statistically significant and quantifiable changes in the numbers of cells being affected. This may be an increase or decrease in the numbers of target cells being attracted within a time period or target area.
The present invention provides reagents which will find use in therapeutic applications as described elsewhere herein, e.g., in the general description for treating disorders associated with skin conditions. See, e.g., Berkow (ed.) The Merck Manual of Diagnosis and Therapy, Merck and Co., Rahway, N.J.; Thorn, et al. Harrison""s Principles of Internal Medicine, McGraw-Hill, NY; Gilman, et al. (eds. 1990) Goodman and Gilman""s: The Pharmacological Bases of Therapeutics, 8th Ed., Pergamon Press; Remington""s Pharmaceutical Sciences, 17th ed. (1990), Mack Publishing Co., Easton, Pa.; Langer (1990) Science 249:1527-1533; and Merck Index, Merck and Co., Rahway, N.J.
Antibodies to MIP-3xcex1 proteins may be used for the identification or sorting of cell populations expressing MIP-3xcex1 protein, e.g., fibroblasts or Langerhans cells. Methods to sort such populations are well known in the art, see, e.g., Melamed, et al. (1990) Flow Cytometry and Sorting Wiley-Liss, Inc., New York, N.Y.; Shapiro (1988) Practical Flow Cytometry Liss, New York, N.Y.; and Robinson, et al. (1993) Handbook of Flow Cytometry Methods Wiley-Liss, New York, N.Y. Populations of cells expressing the MIP-3xcex1 receptor, e.g., CCR6,; can also be purified, e.g., using magnetic beads as described, e.g., in Bieva, et al. (1989) Exp. Hematol. 17:914-920; Hernebtub, et al. (1990) Bioconj. Chem. 1:411-418; Vaccaro (1990) Am. Biotechnol. Lab. 3:30.
Moreover, antisense nucleic acids may be used. For example, antisense polynucleotides against the ligand encoding nucleic acids may function in a manner like ligand antagonists, and antisense against the receptor may function like receptor antagonists. Thus, it may be possible to block the signaling through the pathway with antisense nucleic acids. Conversely, nucleic acids for the receptor may serve as agonists, increasing the numbers of receptor on the cell, thereby increasing cell sensitivity to ligand, and perhaps blocking the normal apoptotic signal described.
Using the assay methods described above, the antibodies or binding compositions are useful in diagnosing diseases states which result in skin disorders. Antibodies raised against a MIP-3xcex1 or CCR6 protein will also be useful to raise anti-idiotypic antibodies. These will be useful in detecting or diagnosing various immunological conditions related to expression of the respective antigens. Combinations of these signals may be also pursued.